Principal Investigator/Program Director(Last, First, Middle): Hough, Loren E. Microtubules (MTs) made from ?- tubulin heterodimers are important for cell migration, long range transport, and cell division. A primary site of tubulin regulation is the C-terminal tails (CTTs). Major questions remain about the molecular mechanism of CTT function and regulation. CTTs affect microtubule length dynamics and mechanical properties even though they contribute only a small percentage of the binding interface between adjacent dimers. CTTs are a major site of tubulin post-translational modi?cation (PTM) which regulates tubulin's binding interactions. PTM of the CTTs alters the processivity of motor proteins1, 2 and the af?nity of proteins which affect MT stability (e.g. MCAK, CLIP-170)3, 4 and proteins which stabilize MTs (e.g. tau).5 Despite their importance, there are few molecular probes of CTT behavior. Because CTTs are ?exible, they are typically undetectable in electron microscopy or x-ray crystallography studies. NMR is the best approach for determining the molecular mechanism of CTT regulation of MT polymerization, mechanics, binding and regulation by PTMs. However, standard techniques for incorporation of heavy isotopes have not worked for tubulin?the proteins have not yet been made in prokaryotic systems, and tubulin typically down regulates its own expression, making over-expression dif?cult in eukaryotic systems. We developed a method to produce heavy isotope labeled tubulin for study by NMR, allowing for a detailed study of the molecular mechanism of CTT function. Building on this breakthrough, we will study the role of CTT polyglycylation in MT regulation. The addition of glycine residues to glutamate side chains occurs on both tubulin CTTs. First identi?ed as one of the two major poly-modi?cations on tubulin, polyglycylation is associated with particularly stable MTs, especially axonemes in mobile cilia.6 Mutations that decrease polyglycylation are associated with reduced stability of cilia, reduced numbers of cilia, increases in cell proliferation, and cancer. However, it is not known why polyglycylation causes these effects. PTMs can operate through a variety of mechanisms: modi?cation of the overall charge distribution,7 alteration of binding interfaces,8 induction of structural changes through allosteric mechanisms,9 and modulation of disordered protein ensembles.10 We will combine NMR, binding assays, molecular simulation, and cell biology to test potential mechanisms of CTT function. We will study two focused questions: 1. How does polyglycylation affect MT polymerization dynamics and stiffness? The presence of the CTT affects MT polymerization dynamics and mechanical properties, suggesting that the CTTs of one dimer interact with other dimers in the MT lattice.11,12 We will purify heavy isotope-labeled tubulin with varying degrees of polyglycylation using mutations in TTLL3-family proteins and mutations in the tubulin CTT at polyglycylation sites. We will use MT assembly assays to determine the effect of polyglycylation on MT nucleation and polymerization dynamics. We will study the effects of polyglycylation on CTT properties by NMR for both dimers and polymer- ized MTs. In conjunction with molecular simulations, we will determine and re?ne hypotheses for the molecular mechanisms by which CTT polyglycylation affects MT properties. We will test these hypotheses through mutation. 2. How does polyglycylation affect MT binding interactions? Removal of the CTTs alters the binding dy- namics of MT-stabilizing and destabilizing binding partners, suggesting that CTT PTM may regulate MTs through effects on MT-interacting proteins. However, little structural information is available on how MT binding partners recognize the CTTs, even those whose interaction is PTM dependent. For example, PTM of the CTTs alters the processivity of motor proteins1, 2 and the af?nity of proteins which affect MT stability (e.g. MCAK, CLIP-170).3, 4 We will determine how the binding af?nity of MT-binding proteins varies with the degree of CTT polyglycyla- tion. By NMR, we will determine the binding interface between the CTT and MT-binding protein, and determine whether and how the binding interface changes when the extent of polyglycylation is altered. We will determine the residues most affected in environment upon binding. This work will allow us to generate and test hypotheses about the molecular mechanism by which poly-G tails affect MT-binding properties. Answering these questions will give unique insight into how tubulin CTT post-translational modi?cation regulates MTs. We are uniquely poised to do this work because we have a direct read-out of CTT average environment and binding interface through the use of NMR. This project will be a detailed characterization of the mechanisms of cellular MT regulation by tubulin CTT polyglycylation and provide a framework for dissecting cellular protein regulation through PTM of disordered domains. Beyond tubulin speci?cally, the T. thermophila heavy labeling, overexpression, and secretion systems will allow our approach to enable NMR structural studies of a wide variety of eukaryotic proteins. Program (Date) Page 0 Proposal Text